Identification and Discrimination of Chlamydia trachomatis Ocular and Urogenital Strains and Major Phylogenetic Lineages by CtGEM Typing, A Double-Locus Genotyping Method.

CtGEM typing was developed to subdivide the bacterial species Chlamydia trachomatis on the basis of genome phylogeny and anatomical tropism. The rationale was facilitation of surveillance for ocular strains, although the method is applicable to essentially any C. trachomatis surveillance application that does not require high resolution. CtGEM is a double-locus genotyping method. The loci included in the assay were identified by computerized analysis of 65 complete genomes for resolution optimized sets of single nucleotide polymorphisms (SNPs). From this, two PCR amplifiable fragments were defined. One, rg1, is within a hypothetical gene annotated as Jali-1891 within the C. trachomatis B_Jali20 genome. The other, ofr, is within the ompA gene which encodes the major outer membrane protein. Variation in rg1 is conferred by two SNPs defining four haplotypes that exhibit concordance with genome phylogeny. Variation within ofr is more complex and allows for inference of ompA genotype, either to the level of single genotype, or group of closely related genotypes. Two CtGEM formats were developed. One is based on interrogation of the two loci by high resolution melting analysis (HRMA), and the other based on analysis of the loci by Sanger sequencing. The genotypes defined identify known ocular genotypes, discriminate known ocular genotypes from each other, discriminate the major phylogenetic lineages of the species, and discriminate all ompA genotypes with the exception of closely related variants within the genotypes H, I, J cluster. The Sanger sequencing format provides slightly more resolution that the HRMA format with respect to ompA genotype. An unusual aspect of this method is that all possible combinations of rg1 haplotype, and inferred ompA genotype(s) have been given CtGEM typing numbers. This includes types that at this time have not been shown to exist.